The present invention relates to isolated cotton leaf curl virus promoters and method for expressing heterologous genes of interest in plant cells by using the promoter.
Plant genetic engineering is aimed to breed transgenic plants that steadily express genes of interest at high level. Gene expression is controlled by the nucleotide sequence within the initiation site of transcription, called promoter elements, and include signals for promotion of transcription by RNA polymerase. Transcription results in the production of messenger RNA, which in turn results in the synthesis of protein.
So far, a variety of promoters derived from various resources have been applied in plant transformation. A main category of which was isolated from Agrobacterium. Another category was derived from plants. The expression levels of genes controlled by these two category of promoters are usually too low or the expression is species/varieties specific, or tissue/organ specific. The most extensively used promoters come from plant viruses. The 35S promoter of cauliflower mosaic virus is one of the most widely used promoters in dicotyledonous plant transformation as it can lead high level expression of heterologous genes in most tissues. Furthermore, in order to enhance the expression of heterologous genes, promoters are usually used in combination with enhancers, cis-elements, non-transcription sequences, intron, exon and 3xe2x80x2 control sequences and the like. For example, the expression level of GUS reporter gene could be enhanced up to 100-fold by using the intron 1 of maize shrunken-1 gene in tobacco cells, and could be enhanced up to 10-fold by using the first exon of maize shrunken-1 gene (C. Mass et al., 1991, xe2x80x9cThe Combination of Novel Stimulatory Element in the First Exon of the Maize Shrunken-1 Gene with the following Intron-1 Enhances Reporter Gene Expression up to 1000-foldxe2x80x9d, Plant Mol. Biol., 16: 199-207).
Promoters from plant geminiviruses could be potentially used in heterologous gene expression. Geminivirus is a bipartite virus with twin particles. There are numerous members of geminivirus and they infect many crops of different kinds. The geminiviruses are classified into three subgroups based on their genome structure and the insect on which they rely for transmission. In subgroup I and II, the geminivirus genomes are mono partite, namely single genome. These two subgroups of geminiviruses are mainly transmitted by leafhopper. The differences between these two subgroups consist in that the former mainly infects monocotyledonous plants while the latter mainly infects dicotyledonous plants. The subgroup III of geminivirues mainly infect dicotyledonous plants and are transmitted by whitefly. Their genome is mostly bipartite, in which the larger one is called DNA A and the smaller one is called DNA B. The genome of some of the subgroup is mono partite.
A prominent characteristic of geminiviruses is that the genomic DNA is circular and single stranded, with a length of about 2400-3000 nucleotides. Another prominent characteristic of geminivirus genome is the bidirectional transcription controlled by the same promoter. The promoter can initiate transcription in two directions, therefore, the promoter is called a bidirectional promoter. One of the two directions controlsexpression of the viral replication protein gene, the other controls the expression of the coat protein gene. Thus, the two directions of the promoter are named the replication protein gene promoter and the coat protein gene promoter, respectively. The geminivirus promoter shares some features of a typical eukaryotic promoter which comprises a TATA box upstream of transcription initiation sites (P. A. Eagle et al., 1997, xe2x80x9cCis-Elements Tthat Contribute to Geminivirus Transcriptional Regulation and the Efficiency of DNA Replicationxe2x80x9d, J. Virol., 71: 6947-6955).
X. Zhan et al. (X. Zhan et al., 1991, xe2x80x9cAnalysis of the Potential Promoter Sequence of African Cassava Mosaic Virus by Transient Expression of the xcex2-Glucuronidase Genexe2x80x9d, J. Gen. Virol., 72: 2849-2852) isolated a bidirectional promoter from African cassava mosaic virus(ACMV) genome which belongs to subgroup III. Transient expression in tobacco protoplast showed that GUS reporter gene expression activity controlled by replication protein gene promoter was 40-fold lower than that of CaMV 35S promoter. The activity of the coat protein gene promoter was very low when used alone. However, the activity can be increased up to 3-fold by the ACMV coded AC2 protein under the control of CaMV 35S promoter (Haley, A. et al., 1992, xe2x80x9cRegulation of African Cassava Mosaic Virus Promoters by the AC1, AC2, AC3 Gene Productsxe2x80x9d, Virology, 188: 905-909).
By transforming a plant using TGMV introduced into Agrobacterium as a gene vector, R. J. Hayes et al. found that the reporter gene activity of neomycin phosphotransferase (NPTII) controlled by coat protein gene promoter from tomato golden mosaic virus(TGMV) activated by AC2 was not as strong as the CaMV 35S promoter, and was 2-fold lower than that of CaMV 35S promoter. (R. J. Hayes et al., 1989, xe2x80x9cReplication of Tomato Golden Mosaic Virus DNA B in Transgenic Plants Expressing Open Reading Frames (ORFs) of DNA A: Requirement of ORF AL2 for Production of Single-Stranded DNAxe2x80x9d, Nucl. Acids Res., 17: 10213-10222).
C. Brough et al. (C. Brough et al., 1992, xe2x80x9cKinetics of Tomato Golden Mosaic Virus DNA Replication and Coat Protein Promoter Activity in Nicotiana Tabacum Protoplastxe2x80x9d, Virology, 187: 1-9) found that transient expression activity of GUS gene controlled by the coat protein gene promoter was up to 2-fold higher than that by the CaMV 35S promoter, which was demonstrated by using non-replicative TGMV vectors for plant transformation. However, the activity of replicative TGMV coat protein gene promoter was about 60-90-fold of that of CaMV 35S promoter.
Xu Yuquan et al. (Y. Xu et al., 1998, xe2x80x9cActivity and Transcriptional Regulation of Bidirectional Promoter from Tobacco Yellow Dwarf Geminivirusxe2x80x9d, Chinese Journal of Virology, 14: 68-74) isolated a subgroup III bidirectional promoter from tobacco yellow dwarf geminivirus, and found that the replication protein gene promoter was stronger and has an activity of 15-20% of the CaMV 35S promoter as demonstrated by transient expression of GUS as a reporter gene in tobacco protoplast and maize cell suspension. The activity of the coat protein gene promoter was weaker but could be increased by C1: C2 protein(corresponding to AC2 protein) by 3-fold.
Most of the previous studies on bidirectional promoters have been carried out by using transient expression. X. Zhan et al. (X. Zhan et al., 1991, xe2x80x9cAnalysis of the Potential Promoter Sequence of African Cassava Mosaic Virus by Transient Expression of the xcex2-Glucuronidase Genexe2x80x9d, J. Gen. Virol., 72: 2849-2852) found that the activity of ACMV replication protein gene promoter was lower than that of the CaMV 35S promoter as demonstrated by Agrobacterium mediated transformation. Briefly, the activities of the presently isolated geminivirus promoters are lower than that of the CaMV 35S promoter. According to this invention, a promoter was isolated from cotton leaf curl virus which belongs to subgroup III of geminivirus and the activity of the promoter was studied by using GUS as the reporter gene.
Cotton Leaf curl virus (CLCuV) is a geminivirus that was found to have infested various crops in Pakistan, Sudan, and India in recent years. According to the statistics for 1993-1994 published by the Pakistan Government, the area of cotton field infested by CLCuV was up to 900,000 hectares and the loss of yield was about 80% (M. Ali et al., 1995, xe2x80x9cCotton Leaf Curl Virus in the Punjab: Current Situation and Review of Workxe2x80x9d, Multan: Central Cotton Research Institute/Ministry of food, Agriculture and Livestock, Government of Pakistan/Asian Development Bank). It has been demonstrated that CLCuV was transmitted by whitefly. The virus affects a wide spectrum of crops, including French bean, okra, tobacco, tomato, cotton and the like. The affected plants show thick leaf veins and yellowish leaves. At present, at least nine varieties of CLCuV have been found. X. Zhou et al. (X. Zhou et al., 1998, Four DNA-A variants among Pakistani isolates of cotton leaf curl virus and their affinities to DNA-A of geminivirus isolates from okra. J. Gen. Virol., 79: 915xcx9c923) studied the evolution of CLCuV based on the DNA sequences of different varieties. Until now no report has been seen on the application of CLCuV bidirectional promoter in plant genetic engineering.
Great progress has been achieved by way of plant genetic engineering in the field of disease-resistant, insect-resistant, herbicide-resistant, stress-resistant, improvement of crop quality, enhancement of the ability of nitrogen fixation, male sterility, delaying maturity and maintaining freshness, changing the flower colors and shapes. However, the expression levels of genes are still low and unstable, hindering the development of plant genetic engineering. Furthermore, most of the presently used promoters are species or variety-specific, organ or tissue-specific. There still exits a need to develop stronger promoters which can be used in heterologous gene expression in plants.
It is an object of the present invention to provide a stronger promoter suitable for transformation of monocotyledonous plants and dicotyledonous plants which can be used for constitutive expression of genes of interest in plants.
The present invention provides a bidirectional promoter derived from a plant virus.
The bidirectional promoter of the present invention constitute a part of the geminivirus genome, the large intergenic region (LIR) in the genome. The LIR comprises a promoter region and regulatory elements for bidirectional transcription, which plays an important role in the replication and bidirectional transcription of the virus.
Specifically, the promoter of the present invention is a part of the cotton leaf curl virus genome. More specifically, the promoter of the present invention has the sequence, SEQ ID NO: 1, shown in FIG. 1.
Preferably, the promoter of the present invention is the full-length promoter element with nucleotides 1-436 of the sequence shown in SEQ ID NO: 1. This promoter can lead to high level expression of heterologous genes in, for example but not limited to, plants and Agrobacterium tumifaciens. This promoter can also be used in combination with the promoters from other sources in the expression of heterologous genes.
The promoter of the present invention can be truncated, nucletides 1-184 of SEQ ID NO: 1. The truncated promoter can lead to high level expression of a heterologous gene in roots as well as in other organs such as stems and leaves of plants. The truncated promoter may also be used in combination with the promoters from other sources in the expression of heterologous genes.
The promoter of the present invention may be a second truncated form, nucleotides 1-278 of SEQ ID NO:1. This second truncated promoter can lead to high level expression of a heterologous gene in, for example, but not limited to, plants and Agrobacterium tumifaciens. This second truncated promoter may also be used in combination with the promoters from other sources in the expression of a heterologous gene.
The promoter of the present invention may further be in a third truncated form, with nucleotides 1-294 of SEQ ID NO:1. This third truncated promoter can lead to a high level expression of a heterologous gene in, for example, but not limited to, plants and Agrobacterium tumifaciens. This third truncated promoter may also be used in combination with the promoters from other sources in the expression of heterologous genes.
The bidirectional promoter of the present invention is derived from the LIR region of the CLCuV genome. The promoter can be isolated from the native CLCuV genome or synthesized by using PCR or any conventional chemical synthesis method. CLCuV belongs to subgroup III and includes a number varieties. At present, nine different strains have been found in Pakistan. In a preferred embodiment of the present invention, the bi-directional promoter is isolated from a new CLCuV strain, and has the sequence illustrated in FIG. 1.
The promoters provided by this invention can be used in either one direction for controlling heterologous gene expression or in both directions at the same time. The promoter may also be used as a combination promoter by linking its controlling elements with other promoter elements. Those skilled in the art will appreciate that, mutation(s) or deletion(s) performed on the bidirectional promoter to the nucleotides beyond the TATA box core region do not affect to a significant extent the activity of the promoter under some circumstances. If desired, the regulatory elements of the bidirectional promoter can be modified before they are used. Therefore, promoters which share 80% identity of the promoter shown in FIG. 1 are also encompassed by the present invention.
Preferably, the promoter provided by this invention includes upstream region of transcription initiation site (position +1), and does not include the region between transcription initiation site and translation initiation site (ATG). The transcription initiation site of an eukaryote is characterized by a sequence of YYYAYA (wherein, Y represents pyrimidine), and transcription starts at the 4th nucleotide which is A. The transcription initiation site of the replication protein gene promoter is at position 2606 which is A, and the transcription initiation site of the coat protein gene promoter is at position 255 which is T, by homology analysis with other known geminivirus genomes.
It is another object of the present invention to provide a method for heterologous gene expression in plants using the promoter of the present invention. The method includes the following steps:
(a) Constructing a plant expression vector by linking the cloned bi-directional promoter from CLCuV with a heterologous gene to be expressed,
(b) Transforming plant cells with the constructed plant expression vector,
(c) Regenerating the transformed plant cells under appropriate culture conditions into plants and identifying and obtaining the transformed plants expressing the heterologous genes.
The promoter used in the method of the present invention is a bidirectional promoter from cotton leaf curl virus. Specifically, the promoter has a sequence illustrated in FIG. 1 or a truncated promoter as mentioned above. The promoter can be isolated from the virus or synthesized chemically.
The promoter used in this method is called bidirectional promoter because the promoter functions in two directions. The gene to be expressed can be linked with the promoter in two different directions.
Preferably, the promoter provided by this invention includes upstream region of transcription initiation site (position +1), and does not include the region between the transcription initiation site and the translation initiation site (ATG). The transcription initiation site of the eukaryote is characterized by a sequence of YYYAYA (SEQ ID NO:4, wherein, Y represents pyrimidine), and the transcription starts at the 4th nucleotide, A. The transcription initiation site of the replication protein gene promoter is at position 2606, A, and transcription initiation site of the coat protein gene promoter is at position 255 T, by homology analysis with other known geminivirus genomes.
Preferably, a promoter useful in this method includes a regulatory element, a cis-element, such as the tissue specific (root-specific, mesophyll-specific, seed endosperm-specific floral organ-specific, fruit-specific, vascular-specific) elements; an inducible element, such as, a wound inducible element; an enhancer element for enhancing heterologous gene expression, or a negative regulatory element for reducing heterologous gene expression as desired. It is known to those skilled in the art that a promoter which lead to constitutive expression is generally composed of a number of modular cis-elements. The presence of regulatory elements determine the characteristics of gene expression, and can be used in combination with a promoter from a different source. The cis-elements of the bidirectional promoter are also included in this invention.
More preferably, the promoter used in this method includes not only the core promoter sequences required for viral gene transcription such as the TATA box, but also includes other cis-elements and elements related to viral replication such as the conserved hairpin and its neighboring sequences, 5xe2x80x2-GCTCCAAAAAGCGGCCATCCGTATAATATTACCGGATGCCGCGCTTT TTTTTTTGTG-3xe2x80x2 (SEQ ID NO:5). Generally, the activity of the promoter would not totally be lost if the sequences beyond the TATA box core region were modified by mutation or deletion. Therefore, if desired, the regulatory elements of the bi-directional promoter can be modified before it is used. Thus, DNA sequences which have approximately 80%, preferably 99%, nucleotide identity with the sequences shown in FIG. 1 are also included in this invention.
The promoter used in this method can include an additional regulatory element (such as an enhancer element) from the CaMV 35S promoter, ocs promoter (L. Comai et al., 1985, xe2x80x9cExpression in Plants of a Mutant AcroA Gene from Salmonella Typhimurium Confers Tolerance to Glyphosatexe2x80x9d, Nature, 317: 741-744), mas promoter (K. E., McBride et al., 1990, xe2x80x9cImproved Binary Vectors for Agrobacterium-Medidated Plant Transformationxe2x80x9d, Plant Mol.Biol., 14: 269-276) to form a combinatory promoter. Moreover, the bidirectional promoter may be placed upstream of any a translation enhancer element to regulate heterologous gene expression. The translation enhancer elements include a non-translation sequence AMV from Alfafa mosaic virus(AMV) (S. A Jobling et al., 1987, xe2x80x9cEnhanced Translation of Chimeric Message RNAs Containing a Plant Viral Untranslated Leader Sequencexe2x80x9d, Nature, 325: 622-625), xcexa9 factor from tobacco mosaic virus (K. Richards et al., 1978, xe2x80x9cNucleotide Sequence at the 5xe2x80x2 Extremity of Tobacco Mosaic Virus RNAxe2x80x9d, Eur. J. Biochem., 84: 513-519) and the like. In the chimeric gene constructs, DNA sequences which can enhance heterologous gene expression can also be included. The DNA sequences include but are not limited to an intron, such as the rice Actin 1 intron, the maize Ubquin intron 1, the maize S-1 intron of ethanol dehydrogenase gene 1, or an exon such as the maize Sh1 exon 1 of sucrose synthetase gene and 3xe2x80x2 regulatory element such as PI-II gene terminator.
The bidirectional promoter in the method of this invention can be linked with a heterologous gene either in the direction of the replication of the protein gene or in the direction of the coat protein gene. The activity of the promoter in the direction of the CLCuV coat protein gene is very low by itself. It can be activated by the AC2 protein encoded by CLCuV genome. Therefore, in the method of this invention, the AC2 protein gene (the DNA sequence is illustrated in FIG. 2) can be co-expressed if the bidirectional promoter were linked with a heterologous gene in the direction of the coat protein gene.
The expression of the AC2 protein gene can be controlled by any of the known promoters, which include, but are not limited to, the CLCuV promoter, the CaMV 35S promoter, the tomato E8 promoter, the Gt3 promoter, rolc promoter, the CoYMV promoter, the rbcs promoter, the hsp70 promoter and the PI-II promoter and the like.
The GUS reporter gene was used in the method of this invention. For the purpose of the present invention, a reporter gene means a nucleic acid sequence fused down stream to the promoter. The promoter can direct transcription of the reporter gene and translation of the reporter gene will produce biochemical substances which can be easily detected by beta-glucuronidase(GUS). In this invention, promoter activity was determined by measuring the activity of beta-glucuronidase in tobacco and cotton cells.
The heterologous gene in this invention can be any of the genes targeted for altering a genetic trait of a plant. These include but not limited to non-coding genes such as RNA genes (include but not limited to sense RNA and anti-sense RNA), and coding genes (include but not limited to genes for insect-resistant, disease-resistant, stress-resistant, herbicide-resistant, improvement of crop quality) which can be used together with the promoter and inserted into vectors and for expression in plant cells.
Gene manupulation can be carried out by know techniques described in, for example, J. Sambrook et al., 1989, xe2x80x9cMolecular Cloning, A Laboratory Manualxe2x80x9d, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Transformation of plants can be performed by the method of, for example, gene gun transformation, leaf-dic Agrobacterium tumifaciens transformation, PEG transformation and the like.
The promoter of the present invention can be used in dicotyledonous plants (include but not limited to cotton, tobacco) for expression of a heterologous gene. It can also be used in monocotyledonous plants, but the expression level of genes controlled by dicotyledonous plant promoters in monocotyledonous plants is not as high. Tobacco is a widely used model plant because it can be easily regenerated and the transformation efficiency is high using Agrobacterium tumifaciens Ti plasmid vector. Dicotyledonous plants such as cotton, tomato, rape, French bean, okra, tobacco and monocotyledonous plants such as grains, for example rice, wheat, maize, barely, oats are preferred hosts for transformation.
The evaluation of gene expression level controlled by the promoter can be carried out by any of the known method in the art, which include, for example, fluorescence spectrophotometry for analysis of GUS, or a histochemical assay, such as the hand cut method or the frozen cut method.
Another subject of the present invention is the use of the AC2 protein encoded by the CLCuV genome to enhance the gene expression level directed by the coat protein gene promoter.
The AC2 protein gene can be found in the total DNA of CLCuV infected plant tissues. It is derived from CLCuV genome and can be obtained by PCR, extraction or chemical synthesis. The amino acid sequence and the corresponding nucleotide sequence are shown in SEQ ID NO: 2 and the deduced amino acid sequence shown in SEQ ID NO:3 in FIG. 2.
The expression of AC2 can be controlled by any of the known promoters able to function in plant cells to regulate the activity of the coat protein gene promoter. Such promoters include, but not limit to, the CLCuV promoter, the CaMV 35S promoter, the tomato E8 promoter, the Gt3 promoter, the rolc promoter, the CoYMV promoter, the rbcs promoter, the hsp7O promoter, and the PI-II promoter.
The promoter of the present invention functions in two directions. The promoter in one direction, the full length replication protein (RP) gene promoter, can direct heterologous gene expression at a level 5.4 times higher than that of the CaMV 35S promoter on average, with the highest level of expression reaching 10 times that of the CaMV 35S promoter. The full-length promoter in this direction can direct heterologous gene expression efficiently in plant roots, stems, and leaf organs, with the highest level in roots.
Histochemical analysis demonstrated that the full length replication protein gene promoter showed activity in mesophyll (including palisade and spongy mesophyll tissues) and vascular tissues. Staining of the stem sections showed that strong GUS staining was detected in both external and internal phloem cells and in the xylem parenchyma cells. In the section prepared from an unfertilized flower, GUS staining was detected in calyx, corolla, ovary, stigma and tapetal. In immature fruits, GUS staining was present on the outside wall. In mature seeds, GUS activity was present in the embryo and the surrounding endosperm tissues.
A heterologous gene can be expressed at a high level in roots under the control of the truncated replication protein gene promoter, nucleotides 1-184 of SEQ ID NO:1. The truncated promoter can also direct heterogeous gene expression at a high level in other organs of plants, such as leaves and stems. In leaves, the expression level directed by this promoter is 2.4 fold higher than that by the CaMV 35S promoter.
A heterologous gene can also be expressed at a high level under the control of another truncated replication protein gene promoter, nucleotides 1-278 of SEQ ID NO:1. In leaves, the expression level directed by this promoter is 6.6 fold higher than that by the CaMV 35S promoter.
A heterologous gene can further be expressed at a high level under the control of another truncated replication protein gene promoter, nucleotides 1-294 of SEQ ID NO:1. In leaves, the expression level directed by this promoter is 10.9 fold higher than that by the CaMV 35S promoter.
Histochemical assay were performed on root, stems and leaves of plants transformed with a vector containing the GUS gene controlled by each of different truncated promoters. The results showed that positions 141-184 contains essential regulatory elements for specific expression in root. The replication protein gene promoter lost its function to direct expression in root without the element represented by positions 141-184. However, this does not exclude the possibility that the promoter without positions 141-184 may direct expression in other tissues.
The activity of the full length (nucleotides 1-436) replication protein gene promoter is 2 times higher than that of the CaMV 35S promoter in Agrobacterium.
The activity in another direction of the promoter, namely the coat protein gene promoter, is very low, 5 times of background (FIG. 4). However, transient expression of genes directed by this promoter can be increased by activation with the AC2 protein (see table 2 below). AC2 gene can act as a switch for gene expression controlling the initiation and termination of transcription directed by the coat protein gene promoter.